run_metadata: 67934
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| 67934 | SRR17499128 | SRX13669342 | SRS11554978 | SRP354077 | PRJNA795663 | Identification of an evolutionarily conserved domain in Neurod1 essential for triggering enteroendocrine cell differentiation | GSE193281 | Transcriptome Analysis | ARP/ASCL transcription factors are key determinants of cell fate specification in a wide variety of tissues coordinating the acquisition of generic cell fates and of specific subtype identities. How these factors recognizing highly similar DNA motifs display specific activities is not yet fully understood. To address this issue we overexpressed different ARP/ASCL factors in zebrafish ascl1a / mutant embryos to determine which one is able to rescue the intestinal secretory lineage. We found that Ascl1a/b Atoh1a/b and Neurod1 factors are all able to trigger the first step of the secretory regulatory cascade but distinct secretory cells are induced by these factors. Indeed Neurod1 rescues the enteroendocrine lineage while Ascl1a/b and Atoh1a/b rescue the goblet cells. Gain of function experiments with Ascl1a/Neurod1 chimeric proteins revealed that the functional divergence is encoded by a 19 aa ultra conserved element UCE present in all Neurod members but absent in the other ARP/ASCL proteins. This novel domain acts as a goblet cell fate repressor and inhibits Gfi1aa expression known to be important for goblet cell differentiation. Deleting the UCE domain of the endogenous Neurod1 protein leads to an increase in the number of goblet cells concomitant with a reduction of several EE subtypes validating the importance of the UCE domain in enteroendocrine cell differentiation. Importantly the neurod1 null mutant displays very similar defects supporting the crucial function of the UCE domain for NeuroD1 activity in the intestine. As Gfi1 acts as a binary cell fate switch in several tissues where Neurod1 is also expressed we can envision a similar role of the UCE in other tissues allowing Neurod1 to repress Gfi1 to influence the balance between cell fates. Overall design: RNA sequencing of 23 samples. Enteroendocrine cell pax6b:GFP + transcriptomic profiles of 4dpf wild type and neurod1 deleted from its conserved domain neurod1DelUCE / zebrafish embryos were generated in in 3 replicates and 4 replicates respectively. Endodermal cells sox17:dsRed + transcriptomic profiles of 52hpf WT or transgenic hsp70:ascl1a hsp70:neurod1 hsp70: neurod1DelUCE hsp70: neurod1DelECD zebrafish embryos were generated in triplicates or in 4 replicates hsp70:neurod1 post 2 heat shocks inducing transgene expression. | pubmed:35286299 | WT 3 | GSM5782115 | source name:Endodermal cells|strain:AB|tissue:Endoderm|age:52hpf|genotype:Wild type|transgene:n1|treatment:heat shock | WT 3 | Sequences were trimmed in order to remove adaptors and low quality bases Trimmed reads were mapped in to the zebrafish genome GRCz11 using STAR software v.2.5.4b Dobin et al. 2013 and gene expression was measured from the mapped reads by using built in STAR module quantMode GeneCounts. Genome build: GRCz11 release 92 Ensembl Supplementary files format and content: tab delimited text files include raw counts for annotations for each Sample | Endodermal cells | The following zebrafish transgenic and mutant lines were used: Tgpax6b:GFPulg515 Delporte et al. 2008b Tgsox17:dsred Field et al 2003 neurod1 ulg052 | EECs were isolated by dissecting the gut from about 200 transgenic neurod1ΔUCE/ΔUCE or wild type Tgpax6b:GFPulg515 larvae at 4 dpf taking care of not including pancreatic tissue. Cell dissociation was performed by incubation in HBSS 1x supplemented with 100 U/ml collagenase IV and 0.3 U/ml Dispase Life Technologies for 10 minutes. Cells were washed in HBSS Mg2+ and Ca2+ free containing 1% BSA and GFP expressing EECs were selected by FACS purification using FACS Aria II. This procedure allows us to obtain between 1000 to 2000 isolated GFP+ cells. Endodermal sox17 dsred+ cells were isolated by dissecting the trunk from about 150 double transgenic Tghsp70l:eGFP 2A ARP/Ascl or wild type Tgsox17:dsred larvae at 52 dpf heat shocked at 38 and 48hpf taking care of not including pancreatic tissue. Cell dissociation was performed by incubation in TrypLE select 1X Gibco for 8 minutes. Cells were washed in HBSS Mg2+ and Ca2+ free containing 1% BSA and dsred+ EECs were selected by FACS purifications using FACS Aria II. This procedure allow us to obtain between 2000 to 4000 isolated dsred+ cells. Each FACS sorted cells sample was directly pelleted by centrifugation and resuspended in 3.5 µl of reaction buffer lysed by freezing in liquid nitrogen and stored at 80°C according the Smart seq2 protocol [49]. cDNA was synthesised and amplified by a 13 cycles PCR reaction. Quality of cDNA was verified by 2100 High Sensitivity DNA assay Agilent technologies. 1 ng cDNA was used for preparing each cDNA library using Nextera XT kit Illumina and sequenced on Hi seq 2000 to obtain around 20 million of reads 75 base single end. | Zebrafish Danio rerio were raised according to standard protocols and staged according to Kimmel Kimmel et al. 1995. | strain:AB|tissue:Endoderm|age:52hpf|genotype:Wild type|transgene:n1|treatment:heat shock | GSM5782115 | GSM5782115: WT 3; Danio rerio; RNA Seq | GSM5782115 r1 | GSM5782115 | 1 | EECs were isolated by dissecting the gut from about 200 transgenic neurod1ΔUCE/ΔUCE or wild type Tgpax6b:GFPulg515 larvae at 4 dpf taking care of not including pancreatic tissue. Cell dissociation was performed by incubation in HBSS 1x supplemented with 100 U/ml collagenase IV and 0.3 U/ml Dispase Life Technologies for 10 minutes. Cells were washed in HBSS Mg2+ and Ca2+ free containing 1% BSA and GFP expressing EECs were selected by FACS purification using FACS Aria II. This procedure allows us to obtain between 1000 to 2000 isolated GFP+ cells. Endodermal sox17 dsred+ cells were isolated by dissecting the trunk from about 150 double transgenic Tghsp70l:eGFP 2A ARP/Ascl or wild type Tgsox17:dsred larvae at 52 dpf heat shocked at 38 and 48hpf taking care of not including pancreatic tissue. Cell dissociation was performed by incubation in TrypLE select 1X Gibco for 8 minutes. Cells were washed in HBSS Mg2+ and Ca2+ free containing 1% BSA and dsred+ EECs were selected by FACS purifications using FACS Aria II. This procedure allow us to obtain between 2000 to 4000 isolated dsred+ cells. Each FACS sorted cells sample was directly pelleted by centrifugation and resuspended in 3.5 µl of reaction buffer lysed by freezing in liquid nitrogen and stored at 80°C according the Smart seq2 protocol [49]. cDNA was synthesised and amplified by a 13 cycles PCR reaction. Quality of cDNA was verified by 2100 High Sensitivity DNA assay Agilent technologies. 1 ng cDNA was used for preparing each cDNA library using Nextera XT kit Illumina and sequenced on Hi seq 2000 to obtain around 20 million of reads 75 base single end. | RNA-Seq | TRANSCRIPTOMIC | cDNA | SINGLE | ILLUMINA | Illumina HiSeq 2000 | SRP354077 | loader:fastq load.py | WT_3.fastq.gz | fastq | 1631087040.0 | 22246950.0 | GSM5782115 r1 | 0:73.32 | A:449912416;C:359801594;G:370071032;T:449082499;N:2219499 | 73 | 449912416 | 359801594 | 370071032 | 449082499 | 2219499 | SRX13669342 | SRS11554978 | SRA1353950 | GIGA, University of Liège | GIGA, University of Liège | 1 | 0.92746 | 0.13827 | 0.7205 | 0.49824 | 76 | B | usable mapping rate | illumina | hiseq_era | unknown | cdna_unspecified | nextera | sc | single_cell_plate | smartseq | Belgium | 2022-01-08 | Hatching | Embryo | Multi-tissue | Multi-system |